Novel anitsense oligonucleotide derivatives against to hepatitis c virus

ABSTRACT

An antisense oligonucleotide derivative against HCV is provided which contains one or more nucleotide analogue units having a modified sugar portion and represented by the following general formula  
                 
where B denotes a pyrimidine or purine nucleic acid base or an analogue thereof. The derivative of the present invention is an antisense against hepatitis C virus (HCV) gene, binds to HCV-RNA with high affinity within cells, can control and inhibit the expression of its gene with high efficiency, and shows high resistance to nucleases. The BNA antisense oligonucleotide of the present invention is also effective in an antisense method targeting HCV, no matter what secondary structures, such as loops or stems, in a target RNA nucleic acid are.

TECHNICAL FIELD

This invention relates to novel antisenses against hepatitis C virus(HCV). More particularly, the invention relates to novel antisensesagainst HCV, which comprise oligonucleotide derivatives containing oneor more nucleotide analogue units having a modified sugar portion.

BACKGROUND ART

In 1978, it was reported for the first time that antisense moleculesinhibited infection by influenza virus. Since then, reports have beenissued that antisense molecules also inhibited oncogene expression andAIDS infection. Since antisense oligonucleotides specifically controlthe expression of undesirable genes, they have become one of the mostpromising fields as medicines in recent years.

The antisense method is based on the concept of inhibiting the processof translation from MRNA into protein in a series of information flowsteps of the so-called central dogma, DNA→mRNA→protein, by use of anantisense oligonucleotide complementary to MRNA, thereby controlling thefunction of the DNA. The antisense method is a method which forms adouble strand with single-stranded mRNA, the cause of disease, with theuse of an antisense oligonucleotide introduced from outside, therebyinhibiting translation into its protein.

When a natural type oligonucleotide was applied as an antisense moleculeto this method, however, the problem arose that it underwent degradationby various nucleases present in vivo, or its cell membrane permeationwas not high. Thus, numerous nucleic acid derivatives or analogues havebeen synthesized, and have been extensively studied. For example,phosphorothioates having an oxygen atom on a phosphorus atom substitutedby a sulfur atom, and methylphosphonates having an oxygen atom on aphosphorus atom substituted by a methyl group were synthesized.Recently, the derivatives or analogues having the phosphorus atom alsosubstituted by a carbon atom, those having the structure of the sugarportion converted, or those having a nucleic acid base modified havealso been synthesized. However, none of these derivatives or analoguesare fully satisfactory in stability within cells, ease of synthesis, andbinding specificity for sequences (the property of selectivelycontrolling only the expression of particular genes).

Japanese Patent Application Laid-Open No. 1998-304889 discloses anucleotide analogue unit modified in the sugar portion of anoligonucleotide constituting an antisense. This nucleotide analogue unithas a structure represented by the following formula 1 in which thesugar conformation is fixed in the N type

In this publication, an oligonucleotide derivative incorporating one ormore nucleotide analogue units having the above-described sugar portionis synthesized, and the fundamental physical properties of theoligonucleotide derivative are measured extracellularly. That is, theoligonucleotide derivative and a sense strand comprising natural DNA orRNA are subjected to annealing, and the melting temperature of theannealing product is measured to investigate double strand formingcapacity. Moreover, the resistance of this oligonucleotide derivative tonuclease enzymes is measured in vitro.

However, this publication does not disclose whether or not theoligonucleotide derivative shows nuclease resistance intracellularly asin the results of extracellular experiments to act stably as anantisense, or whether or not the oligonucleotide derivative binds to anaturally occurring gene particularly within cells to form a doublestrand or a triple strand, thereby becoming able to inhibit theexpression of a particular gene actually.

The discovery of hepatitis C virus (HCV), on the other hand, isrelatively recent, and a gene of HCV was isolated in 1988 (Choo, Q, L.et al., Science, 244, 359-362 (1989)). In connection with HCV, manyreports have been made of an entire length base sequence and an aminoacid sequence, a 5′-untranslated region, an internal ribosomal entrysite (IRES), a stem region, etc. (Kato. N. et al., Proc. Natl. Acad.Sci. USA., 87, 9524-9528, (1990), Proc. Natl. Acad. Sci. USA., 88,2451-2455, (1991), J. Virol., 65, 1105-1113, (1991), J. Gen. Virol., 72,2697-2704, (1991), Virology, 188, 331-341, (1992), Tsukiyama. Kohara. etal., J. Virol., 66, 1476-1483, (1992), S. Tang. et al., J. Virol., 73,2359-2364, (1999), Honda Masao et al., J. Virol., 73, 1165-1174, (1999),Honda Masao et al., RNA., 2(10), 955-968, (1996), Sasano T. et al.,Genome Inf. Ser., 9, 395-396, (1998), Ito T. et al., J. Virol., 72,8789-8796, (1998), Kamoshita N. et al., Virology., 233, 9-18, (1997),etc.). For HCV, interferon (IFN) has been said to be the only therapy.However, the severity of its side effects and its low rate of markedefficacy have been mentioned as major problems with treatment using IFN.Some reports say that IFN is effective in only 20% of patients. Toincrease its effectiveness, attempts such as a combination therapy withIFN and ribavirin have been applied. However, such attempts have notproduced satisfactory results.

Aiming for gene therapy, techniques using the antisense method,ribozyme, and DNAzyme have been investigated, and in vivo studies on theantisense method and ribozyme are under way. Techniques concerned withantisenses, in particular, have widely propagated, and many reports onantisenses against HCV have been presented (WO 99/29350, WO 99/29350, WO95/30746, WO 94/08002, Wakita et al., J. Biol. Chem., 269, 14205-14210,(1994), WO 94/24864). However, few antisenses satisfactory in enzymeresistance, affinity, and cytotoxicity have been obtained.

For the purpose of use in such an antisense method, it is desired tocreate an antisense oligonucleotide derivative, especially an antisenseoligonucleotide derivative against HCV, which is minimally degraded bynucleases within cells, and binds to targeted HCV with high affinity,thus being capable of controlling and inhibiting, with high efficiency,the expression of its gene.

DISCLOSURE OF THE INVENTION

We, the inventors of the present invention, designed a nucleic acidanalogue, in which the conformation of a sugar portion in the nucleicacid has been fixed, as a technique considered to be useful in theantisense method. We synthesized a nucleotide analogue unit serving as aunit structure for the nucleic acid analogue, and prepared anoligonucleotide derivative with the use of the nucleotide analogue unit.We have confirmed that this oligonucleotide derivative is very useful asan HCV antisense molecule.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 is a view showing the structure of an HCV genome.

FIG. 2 is an illustrative view showing the secondary structures ofHCV-IRES.

FIG. 3 is an explanatory view showing the method of constructing atarget plasmid containing HCV-IRES.

FIG. 4 is an explanatory view showing the method of constructing thetarget plasmid containing HCV-IRES (SEQ ID NO:5 and SEQ ID NO:6).

FIG. 5 is an explanatory view showing the method of constructing thetarget plasmid containing HCV-IRES.

FIG. 6 is a graph showing the HCV gene translation inhibiting effect ofan antisense oligonucleotide derivative according to the presentinvention and a natural type antisense oligonucleotide which target thevicinity of the start codon of HCV-IRES in a non-cell system.

FIG. 7 is a graph showing the HCV gene translation inhibiting effect ofan antisense oligonucleotide derivative according to the presentinvention and a natural type antisense oligonucleotide (different inlength from those of FIG. 6) in a non-cell system.

FIG. 8 is a graph showing the HCV gene translation inhibiting effect ofan antisense oligonucleotide derivative according to the presentinvention and a natural type antisense oligonucleotide (different inlength from those of FIGS. 6 and 7) in a non-cell system.

FIG. 9 is a graph showing the HCV gene translation inhibiting effect ofan antisense oligonucleotide derivative according to the presentinvention and a natural type antisense oligonucleotide which target thestem region of HCV-IRES in a non-cell system.

FIG. 10 is a view showing the structures of plasmids used in tests forinvestigating the HCV gene translation inhibiting effect of an antisenseoligonucleotide in a cell system of Example 3.

FIG. 11 is a graph showing the HCV gene translation inhibiting effect ofan antisense oligonucleotide derivative according to the presentinvention and a natural type antisense oligonucleotide, etc. whichtarget the vicinity of the start codon of HCV-IRES in a cell system.

FIG. 12 is a graph showing the HCV gene translation inhibiting effect ofan antisense oligonucleotide derivative according to the presentinvention (D-A159) and a natural type antisense oligonucleotide(BNA-A159) which target the stem region of HCV-IRES in a cell system.

FIG. 13 is a graph showing the HCV gene translation inhibiting effect ofan antisense oligonucleotide derivative according to the presentinvention (BNA-A191) and a natural type antisense oligonucleotide(D-A191) which target the stem region of HCV-IRES in a cell system.

HCV is a single-stranded RNA virus, whose genome is about 9,500 bases inentire length, and which has one open reading frame in its inside (FIG.4). This HCV-RNA is characterized in that translation starts in acap-independent manner. For this purpose, it is required that ribosomesbe bound to IRES (internal ribosomal entry site) within the 5′untranslated region (5′-UTR). This HCV-IRES is a highly conserved regionhaving secondary structures as shown in FIG. 2, and has importantfunctions. This is a region selected in the present invention as one oftargets of the antisense method.

Thus, we synthesized antisense oligonucleotide derivatives, whilesetting, as an actual target, the vicinity of the start codon withinIRES including the 5′ untranslated region selected as a target of theantisense method against HCV. We confirmed these derivatives to have anexcellent binding affinity and a sequence-specific marvelous antisenseeffect, and accomplished the present invention.

We also synthesized antisense oligonucleotide derivatives according tothe present invention, targeted at a plurality of stem regions withinHCV-IRES for which natural type oligonucleotides have shown no antisenseeffect. We confirmed the binding affinity and antisense effect of thisderivative, and accomplished the present invention.

EMBODIMENTS OF THE INVENTION

The oligonucleotide or polynucleotide derivative of the presentinvention has one or more nucleotide analogue units having a structurehaving a sugar conformation fixed, which is represented by the followinggeneral formula

where B denotes a pyrimidine nucleic acid base or a purine nucleic acidbase or an analogue thereof. Hereinafter, such an oligonucleotidederivative or a polynucleotide derivative will be referred to as a BNAoligonucleotide.

The oligonucleotide or polynucleotide derivative of the presentinvention is an oligonucleotide or polynucleotide derivative representedby the following general formula

where B¹ and B² are the same or different, and each denote a pyrimidinenucleic acid base or a purine nucleic acid base or an analogue thereof;R represents hydrogen, a hydroxyl group, halogen, or an alkoxy group; W¹and W² are the same or different, and each denote a natural typenucleotide, a synthetic nucleotide, or an oligonucleotide orpolynucleotide, including any of these nucleotides, mediated byhydrogen, an alkyl group, an alkenyl group, an alkinyl group, acycloalkyl group, an aralkyl group, an aryl group, an acyl group, asilyl group, a phosphate residue or a phosphodiester bond; n¹ or n² isthe same or different, and denotes an integer of 0 to 50, provided thatn¹ or n² is not zero at the same time and that all of n²'s are not zeroat the same time; and n³ denotes an integer of 1 to 50, provided thatwhen n¹ and/or n² are or is 2 or more, B¹ and B need not be the same,and R's need not be the same.

There is no limitation on the number of the bases of the antisenseoligonucleotide derivative according to the present invention, but thisnumber is usually 5 to 50, preferably 9 to 30.

The pyrimidine nucleic acid base or purine nucleic acid base or analoguethereof in the present invention refers to thymine, uracil, cytosine,adenine, guanine or an analogue of any of these. As the analogues,purine nucleic acid base analogues and pyrimidine nucleic acid analoguescan be named.

The purine nucleic acid base analogues can be selected preferably fromthe following compounds:

Guanosine diphosphate, 8-oxo-adenosine, 8-oxo-guanosine,8-fluoro-adenosine, 8-fluoro-guanosine, 8-methoxy-adenosine,8-methoxy-guanosine, 8-aza-adenosine, 8-aza-guanosine, azacytidine,fludarabine phosphate, 6-MP, 6-TG, azathioprine, allopurinol, acyclovir,ganciclovir, deoxyformicin, arabinosyladenine (ara-A), guanosinediphosphate-fucose, guanosine diphosphate-2-fluorofucose, guanosinediphosphate-βL-2-aminofucose, guanosine diphosphate-D-arabinose, and2-aminoadenine.

The pyridine nucleic acid base analogues can be selected preferably fromthe following compounds:

5-fluorouracil, 5-chlorouracil, 5-bromouracil, dihydrouracil,5-methylcytosine, 5-propynylthymine, 5-propynyluracil,5-propynylcytosine, 5-fluorocytosine, fluoxyuridine, uridine, thymine,3′-azido-deoxythymidine, 2-fluorodeoxycytidine,3-fluoro-3′-deoxythymidine, 3′-dideoxycytidin-2′-ene,3′-deoxy-3′-deoxythymidin-2′-ene, and cytosine arabinose.

The synthesis of the nucleotide analogue unit usable as an antisense inthe present invention, and the synthesis of the oligonucleotidederivative containing the nucleotide analogue unit are described indetail in the aforementioned Japanese Patent Application Laid-Open No.1998-304889. These descriptions are to be included herein.

The antisense oligonucleotide derivative against HCV in the presentinvention may be any oligo- or polynucleotide, if it can bind to HCV,inhibiting or suppressing the expression of its gene. It suffices thatone or more of its nucleotides, its constituent units, are substitutedby the nucleotide analogue units of the present invention. Examples ofthe sequence of the antisense binding to HCV are sequences complementaryto the base sequence of HCV, and sequences which hybridize with the basesequence of HCV under stringent conditions.

Examples of the antisense oligonucleotide derivative are those againstthe vicinity of the start codon within IRES including the 5′untranslated region of HCV-RNA, and those against the stem region ofHCV-RNA. Others which target various regions permitting the object ofthe present to be attained can be used. For example, those against exonor those against intron are named. More concretely, the followingantisense base sequences against HCV are included:

(Sequences targeting the vicinity of the translation start codon withinIRES) CTTTGAGGTTTAGGATTCGTGCTCATG (A367) (SEQ ID NO: 1)AGGTTTAGGATTCGTGCTCATG (A362) (SEQ ID NO: 2) TAGGATTCGTGCTCATG (A357)(SEQ ID NO: 3)

(Sequences targeting the stem region within IRES) CCGGTTCCGCAGACCACTAT(A159) (SEQ ID NO: 4) ACCCGGTCGTCCTGGCAATT (A191) (SEQ ID NO: 11)

As the antisense against HCV according to the present invention, therecan be named sequences in which one or more bases of the antisensesequences against HCV represented by the above sequences have beensubstituted by the nucleotide analogue units of the present invention;antisense oligonucleotide derivatives containing these sequences; andoligonucleotide derivatives which hybridize under stringent conditionswith DNA or RNA as a sequence complementary to the above antisensesequences. Hybridization is a well known technique (Sambrook, J. et al.,Molecular Cloning 2nd ed., Cold Spring Harbor Lab. Press, 1989, etc.).

The stringent conditions, referred to in the present invention, can beselected, as desired, by people skilled in the art. An example is lowstringent conditions. The low stringent conditions refer, for example,to 42° C., 0.1×SSC, 0.1% SDS, preferably 50° C., 0.1×SSC, 0.1% SDS, inwashing after hybridization. More preferred stringent conditions arehigh stringent conditions. The high stringent conditions include 65° C.,6×SSC, 0.1% SDS.

The oligonucleotide derivatives of the present invention have also beenconfirmed to show excellent nuclease resistance as compared with naturaltype oligonucleotides having the same base sequence. Thus, theoligonucleotide derivatives of the present invention are advantageous inthat even when administered into cells, they are stable, and theirexcellent antisense effect persists (T. Imanishi, S. Obika, J. Synth.Org. Chem., 11, 969 (1999)).

The antisense oligonucleotide derivatives of the present inventionessentially require the use of the nucleotide analogue unit having onlythe sugar portion modified. It is permissible to use the nucleotideanalogue unit in which in addition to modification of the sugar portion,other portion, for example, the phosphodiester portion has beenmodified, as in phosphorothioates. It is also possible to constructoligonucleotide derivative antisenses by combining the nucleotideanalogue unit of the present invention having a modified sugar portionwith other nucleotide analogue units as publicly known antisenseconstituent units.

The antisense oligonucleotide derivatives of the present invention canbe mixed with suitable base materials inert thereto, wherebypreparations for external use, such as liniments and cataplasms, can beformed. If desired, vehicles, tonicity agents, solution adjuvants,stabilizers, preservatives, and soothing agents are added to theantisense oligonucleotide derivatives, whereby tablets, powders,granules, capsules, liposome capsules, injections, liquids andsolutions, nasal drops, and lyophilized products can be formed. Thesepreparations can be produced by the usual methods.

The antisense oligonucleotide derivatives of the present invention aredirectly administered to the lesion of the patient, or administered intothe blood vessel so that they can eventually reach the lesion.Furthermore, antisense encapsulation materials for enhancing prolongedaction or membrane permeation can also be used. For example, liposome,poly-L-lysine, lipid, cholesterol, lipofectin or an analogue of any ofthese can be named.

The dose of the antisense oligonucleotide derivative of the presentinvention may be a preferred amount adjusted, as desired, depending onthe condition, age, sex and body weight of the patient. The mode ofadministration may depend on the condition of the patient, the dosageform, etc., and may be a preferred method selected, as desired, fromvarious methods of administration, such as oral administration,intramuscular administration, intraperitoneal administration,intradermal administration, subcutaneous administration, intravenousadministration, intraarterial administration, and rectal administration.

The antisense oligonucleotide derivatives against HCV according to thepresent invention show various excellent characteristics as antisenseDNA's. This will be described in detail by the following Examples.

Example 1: Double strand forming capacity of oligonucleotide derivativescontaining nucleotide analogue unit of the present invention bymeasurement of melting temperature

(1) With the vicinity of the start codon (342 nt-344 nt) within HCV-IRESbeing contemplated as a target, antisense oligonucleotides having thesequences indicated below were designed and constructed. To investigatethe relation between the length of the antisense oligonucleotide and theantisense effect, 5 bases and 10 bases were cut off from oligonucleotideA-367, beginning at the 5′-terminal, to synthesize A362 and A357,respectively. CTTTGAGGTTTAGGATTCGTGCTCATG (A367) (SEQ ID NO: 1)AGGTTTAGGATTCGTGCTCATG (A362) (SEQ ID NO: 2) TAGGATTCGTGCTCATG (A357)(SEQ ID NO: 3)

In connection with these oligonucleotides of various lengths, moreover,BNA oligonucleotides (BNA-oligo) and phosphorothioate typeoligonucleotides (S-oligo) were prepared in addition to natural typeoligonucleotides (D-oligo). In regard to A367, for example, D-A367(natural type), BNA-A367 (BNA type) and S-A367 (phosphorothioate type)were prepared.

The sequences of the synthesized oligonucleotides were as follows:5′-CTTTGAGGTTTAGGATTCGTGCTCATG-3′ (D-A367) (SEQ ID NO: 1)5′-CTTTGAGGTTTAGGATTCGTGCTCATG-3′ (BNA-A367) (SEQ ID NO: 1)5′-ctttgaggtttaggattcgtgctcatg-3′ (S-A367) (SEQ ID NO: 1)5′-AGGTTTAGGATTCGTGCTCATG-3′ (D-A362) (SEQ ID NO: 2)

5′-AGGTTTAGGATTCGTGCTCATG-3′ (BNA-A362) (SEQ ID NO: 2)5′-aggtttaggattcgtgctcatg-3′ (S-A362) (SEQ ID NO: 2)5′-TAGGATTCGTGCTCATG-3′ (D-A357) (SEQ ID NO: 3) 5′-TAGGATTCGTGCTCATG-3′(BNA-A357) (SEQ ID NO: 3)

(2) To investigate the binding affinity of these synthesized antisenseoligonucleotides for complementary strand RNA, their Tm values (meltingtemperatures) were measured.

The above synthesized natural type oligonucleotides and oligonucleotidederivatives were used as antisense strands, and annealed to sensestrands comprising natural type DNA or RNA. The melting temperatures (Tmvalues) of the annealing products were measured to examine thehybridizing capacity of the oligonucleotide derivatives of the presentinvention for complementary DNA and RNA.

A sample solution (500 μl) with end concentrations of NaCl 100 mM,sodium phosphate buffer (pH 7.2) 10 mM, antisense strand 1 μM, and sensestrand 1 μM was bathed in boiling water, and cooled slowly to roomtemperature over 10 hours. While a nitrogen stream was passed into acell chamber of a spectrophotometer (Beckman DU650) for prevention ofdew formation, the sample solution was gradually cooled to 5° C., andfurther held at 5° C. for 20 minutes, whereafter measurement wasstarted. The temperature of the sample was raised to 95° C. at a rate of0.5° C./minute, and ultraviolet absorption at 260 nm was measured atintervals of 0.5° C. The intensity of the ultraviolet absorption greatlyincreased at particular temperatures. The temperature at the mid-pointof this transition was the melting temperature, i.e. Tm value. Thehigher the binding affinity, the higher the Tm value.

The results are shown in the following table. In the nucleotidesequences, the nucleotide analogue units of the present invention areunderlined. TABLE 1 Melting temperatures (Tm values) of antisenseoligonucleotide derivatives hybridized with complementary RNA's Tm value(ΔTm/modification)/° C. 100 mM 10 mM 0 mM antisense oligonucleotidesNaCl NaCl NaCl 5′-CTTTGAGGTTTAGGATTCGTGCTCATG-3′ 66 54 50 (D-A367) (SEQID NO: 1) 5′-CTTTGAGGTTTAGGATTCGTGCTCATG-3′ >95 79 (+2.3) 75 (+2.3)(BNA-A367) (SEQ ID NO: 1) 5′-ctttgaggtttaggattcgtgctcatg-3′ 57 44 41(S-A367) (SEQ ID NO: 1) 5′-AGGTTTAGGATTCGTGCTCATG-3′ 64 52 48 (D-A362)(SEQ ID NO: 2) 5′-AGGTTTAGGATTCGTGCTCATG-3′ 88 (+3.0) 76 (+3.0) 71(+2.9) (BNA-A362) (SEQ ID NO: 2) 5′-aggtttaggattcgtgctcatg-3′ 54 43 39(S-A362) (SEQ ID NO: 2) 5′-TAGGATTCGTGCTCATG-3′ 59 48 44 (D-A357) (SEQID NO: 3) 5′-TAGGATTCGTGCTCATG-3′ 79 (+3.3) 67 (+3.2) 63 (+3.2)(BNA-A357) (SEQ ID NO: 3)Conditions; 20 mM sodium phosphate buffer, pH7.2, conc. = 1 μM for allstrandTarget RNA 5′-CAUGAGCACGAAUCCUAAACCUCAAAG-3′ (SEQ ID NO: 13)

(3) Natural type antisense oligonucleotide (D-oligo) and theoligonucleotide derivative (BNA-oligo) of the present invention,targeting the stem region within HCV-IRES, were synthesized in the samemanner as in (1) stated above. In the nucleotide sequences of thefollowing formulas, the nucleotide analogue units of the presentinvention are underlined. CCGGTTCCGCAGACCACTAT (D-A159) (SEQ ID NO: 4)CCGGTTCCGCAGACCACTAT (BNA-A159) (SEQ ID NO: 4)

The Tm values were measured under the same conditions as for theabove-mentioned antisense oligonucleotides targeting HCV-IRES.

The results are shown in Table 2 below. TABLE 2 Tm value (ΔTm/mod.)/° C.Test Antisense molecule 100 mM NaCl 0 mM NaCl 1. D-A159 69 54 2.BNA-A159 >95 84 (+3.0)

As clear from Table 1 and Table 2, the s-oligo's showed decreases in theTm values, i.e. decline of binding affinity, in comparison with theD-oligo's, while the BNA-oligo's showed marked increases in the Tmvalues. These findings confirmed that the BNA-oligo's had very highbinding affinity for complementary strand RNA as compared with theS-oligo's and the D-oligo's. This outcome was confirmed in the case ofthe start codon target and the stem region target. In connection withthe start codon target, the same results were noted for theoligonucleotides with decreased lengths.

Example 2: HCV gene translation inhibiting effect of oligonucleotidederivatives in non-cell system

In this Example, the antisense effect against HCV gene in a non-cellsystem was examined. Target plasmid pcDNA3-IES/Luc containing IRES(internal ribosomal entry site) within the 5′ untranslated region ofhepatitis C virus was designed and constructed. This target plasmid wasused to check for the antisense effect of oligonucleotide derivatives.

(Construction of Plasmid pcDNA3-IES/Luc)

In accordance with the method of Caselmann (Caselmann et al.,Hepatology, 22, 707 (1995)), BK157 (A. Takamizawa et al., J. Virol., 65,1105 (1991)), a clone DNA of HCV, was digested with KpnI and AatII toprepare a fragment A containing the target site (see FIG. 3).

Then, a fragment B containing a luciferase gene (Luc) to be used as areporter gene was prepared (see FIG. 4). This step was performed byamplifying Luc by PCR of pTRE-Luc with the use of a primer A and aprimer B, and then digesting the amplification product with BamHI andXhoI.

As a problem with incorporation of these fragments A and B into a pcDNA3vector, it was expected that the incorporation of the fragment B wouldnot be performed, since many recognition sequences for AatII are presentin pcDNA. Thus, it was planned that both the fragments A and B would beincorporated into a pCRII vector, and then the vector would be replacedby pcDNA3 at the final stage to obtain the desired plasmid (see FIG. 9).

First, a pCRII vector (a product of Invitrogen) was digested with BamHIand XhoI, and then the digest was ligated to the fragment B containingthe luciferase gene, thereby obtaining pCRII-Luc. The resultingpCRII-Luc was digested with KpnI and XhoI, and the digest was ligated tothe fragment A containing the target site, thereby obtainingpCRII-IRES/Luc.

Finally, in order to replace the vector of pCRII-IRES/Luc by pcDNA3having promoters (promoters CMV and T7) capable of acting in both of anon-cell system and a cell system, the pCRII-IRES/Luc and a pcDNA3vector (a product of Invitrogen) were digested with KpnI and XhoI. Then,the digests were ligated together, successfully preparing the desiredtarget plasmid pcDNA3-IRES/Luc. Using this plasmid, the antisense effectagainst HCV was evaluated by the following experiments:

(HCV gene translation inhibiting effect in non-cell system)

(1) In expectation of an increase in the efficiency of in vivotranscription reaction of the target plasmid pcDNA3-IES/Luc containingIRES (internal ribosomal entry site) within the 5′ untranslated regionof hepatitis C virus, the restriction enzyme XhoI was used forlinearization. The resulting linearized DNA was used for in vitrotranscription reaction to synthesize mRNA. This mRNA was converted intoa protein by an in vitro translation reaction using an oligonucleotideand a rabbit reticulocyte solution (RRL), and then luciferase assay wasperformed.

Initially, a study was conducted using an antisense oligonucleotidetargeting the vicinity of the start codon. In this study, D-oligo andBNA-oligo were used. The antisense oligo's (D-A367, BNA-A367) forD-oligo and BNA-oligo, and random oligo's (D-R367, BNA-R367) as negativecontrols were each added such that the ratio of their concentration tothe concentration of mRNA would be 2.5:1, 5:1 and 10:1, whereafter atranslation reaction was carried out. The resulting protein was used forluciferase assay. The results are shown in FIG. 6, with the outcome forthe translation reaction performed without addition of theoligonucleotide being taken as 100%.

The results confirmed that the expression of luciferase was suppressedby the addition of the antisense oligonucleotide, and this suppressionof expression was augmented as the concentration of the oligonucleotideadded was increased, thus showing a concentration-dependent antisenseeffect.

The antisense effect was observed in each of D-A367 and BNA-A367.However, BNA-A367 showed a better effect of suppressing gene expressionat any of the concentrations, and proved to be a better antisenseoligonucleotide. No gene expression suppressing effect was observed inthe random oligonucleotide (BNA-R367) used as the control. Thus, thisantisense effect was confirmed to be sequence-specific.

(2) Then, in order to examine changes in the antisense effect bydecreasing the length of the oligonucleotide, the same tests asdescribed above were conducted using the D-oligo and BNA-oligo of eachof A362 which was decreased by 5 bases from A-367 at its 5′ side, andA357 which was decreased by 10 bases from A-367 at its 5′ side.

The results with the respective oligonucleotides are shown in FIGS. 7and 8, with the outcome for the translation reaction performed withoutaddition of the oligonucleotide being taken as 100%.

The results shown in FIGS. 7 and 8 confirmed that the BNA antisenseoligonucleotides showed an excellent gene expression suppressing effect,even though they had decreased lengths. Particularly, even at the lowconcentrations at which the D-oligo's did not show a high antisenseeffect, the BNA-oligo's were confirmed to produce a significantantisense effect. Neither BNA-R362 nor BNA-R357 showed a gene expressionsuppressing effect. Thus, the antisense effect was confirmed to besequence-specific.

(3) The same tests were conducted on D-A159 and BNA-A159 targeting thestem region within HCV-RNA. Each oligonucleotide was added such that theratio of its concentration to the concentration of MRNA would be 5:1,10:1 and 20:1, whereafter a translation reaction was performed. Theresults of luciferase assay are shown in FIG. 9, with the outcome forthe translation reaction performed without addition of theoligonucleotide being taken as 100%.

When D-A159 was used, no gene expression suppressing effect was observedat any of the concentrations. The reason may be that the binding forceof the D-oligo was too weak to strip off the intramolecular hydrogenbond of the stem region for binding to the stem region.

When BNA-A195 was used, on the other hand, it showed a gene expressionsuppressing effect of about 50% when its ratio to mRNA was 10:1 and20:1. This may be attributed to the potent binding affinity of the BNAoligonucleotide of the present invention taking effect.

Example 3 HCV gene translation inhibiting effect of BNA antisenseoligonucleotide in cell system

The HCV gene translation inhibiting effect was investigated in a cellsystem with the use of HepG2 cells (provided by Osaka University Facultyof Pharmacy), a cell strain of the hepatic cancer origin.

(1) HepG2 cells were seeded onto each well in the same amount of 5×10⁴cells, and incubated for 24 hours. Then, an oligonucleotide, the targetplasmid pcDNA3-IRES/Luc, and pRL-TK were transfected using a lipofectamine in the absence of serum. The pRL-TK, transfected together with thetarget plasmid, is a plasmid free from HCV-IRES, the target site. Thisplasmid was used to investigate the toxicity of the oligonucleotideadded, namely, its nonspecific expression suppressing effect. The pRL-TKcontains the luciferase gene of a sea pansy (renilla) as a reportergene. By performing dual luciferase assay, the PRL-TK enables assay tobe performed separately from the luciferase gene of a firefly, which isthe reporter gene of the target plasmid pcDNA3-IRES/Luc (FIG. 10). Thatis, the one which suppresses the expression of renilla luciferase freeof the target shows a nonspecific gene expression suppressing effect.

Transfection in Opti-MEM was performed for 4 hours in the absence ofserum, and then the culture medium was replaced by DMEM where serum waspresent. Then, incubation was performed for 20 hours, and the cells wereharvested and subjected to dual luciferase assay.

(2) First, a study was made of antisense oligonucleotides targeting thestart codon. The oligonucleotides used in tests were 27 mer antisensesand random oligonucleotides for D-oligo, BNA-oligo and S-oligo, whichwere added at end concentrations of 120 nM.

The results are shown in FIG. 11, with the outcome for transfection ofthe plasmid performed without addition of the oligonucleotide beingtaken as 100%.

As shown in the results of FIG. 11, D-A367, the D-oligo that produced anantisense effect in the non-cell system, showed no gene expressionsuppressing effect in this test in the cell system. This may be becausethe D-oligo was degraded by nucleases present in the cells. Inconnection with the S-oligo evaluated to have excellent enzymeresistance, the expression of firefly luciferase was markedly suppressedby S-367. However, the S-oligo poses the problem of toxicity, and wasthus shown to have a nonspecific expression suppressing effect. That is,the suppression of firefly luciferase expression was observed in therandom oligonucleotide S-R367 which theoretically does not suppressexpression. Additionally, S-A367 and S-R367 also suppress the expressionof renilla luciferase free of the target sequence.

In the case of the BNA oligonucleotide, BNA-A367 showed an excellenteffect of suppressing the gene expression of firefly luciferase, and didnot affect the expression of renilla luciferase. The randomoligonucleotide BNA-R367 showed no gene expression suppressing effect.In the light of these results, it is believed that the effect of the BNAantisense oligonucleotide is a sequence-specific effect, which isascribed to the excellent binding affinity and enzyme resistance of theBNA oligonucleotide. Thus, it has become clear that only the BNAoligonucleotide shows an excellent antisense effect even within cells.

(3) Next, a study was made of the intracellular antisense effect ofBNA-A159 targeting the stem region within HCV-IRES. The oligonucleotidesused were D-A159 and BNA-A159, and they were transfected at endconcentrations of 60 nM and 120 nM. The results are shown in FIG. 12,with the outcome for transfection performed without addition of theoligonucleotide being taken as 100%.

As shown in the results of FIG. 12, D-A159 showed no expressionsuppressing effect as in the non-cell system, and was confirmed toproduce no antisense effect. BNA-A159, on the other hand, producedsuppression of about 50% at 60 nM, and nearly completely suppressed theexpression of luciferase at a concentration of 120 nM. Thus, BNA-A159was confirmed to show an excellent antisense effect.

(4) A natural type antisense oligonucleotide (D-oligo) and theoligonucleotide derivative of the present invention (BNA-oligo) havingthe following sequences, which target the stem region within HCV-IRES,were synthesized in the same manner as for D-A159 and BNA-A159. In thenucleotide sequences of the following formulas, the nucleotide analogueunits of the present invention are underlined. ACCCGGTCGTCCTGGCAATT(D-A191) (SEQ ID NO: 11) ACCCGGTCGTCCTGGCAATT (BNA-A191) (SEQ ID NO: 11)

The intracellular antisense effect of BNA-A191 was studied similarly toBNA-A159. D-A191 and BNA-A191 were used and transfected at endconcentrations of 60 nM, 120 nM and 240 nM. The results are shown inFIG. 13, with the outcome for transfection performed without addition ofthe oligonucleotide being taken as 100%.

As shown in the results of FIG. 13, D-191 showed no expressionsuppressing effect, and was confirmed to produce no antisense effect.

BNA-A191, on the other hand, was confirmed to show an excellentantisense effect.

The above remarkable effect of the BNA antisense oligonucleotide in thecell system as compared with the non-cell system is presumed to beascribed to its excellent enzyme resistance and the high intracellularconcentration of ribonucleases.

INDUSTRIAL APPLICABILITY

The BNA antisense oligonucleotides of the present invention haveexcellent binding affinity for HCV-RNA, show high enzyme resistance, andlow in cytotoxicity. Thus, they show a specific HCV gene expressionsuppressing effect in both of a non-cell system and a cell system. TheBNA antisense oligonucleotides of the present invention are alsoeffective in an antisense method targeting HCV, no matter what secondarystructures, such as loops or stems, in a target RNA nucleic acid are.Hence, the BNA antisense oligonucleotides of the present invention arepromising as therapeutics for gene therapy of HCV for which there havebeen no potent therapeutic drugs except interferon.

1. An antisense oligonucleotide derivative against hepatitis C virus,which contains one or more nucleotide analogue units having a modifiedsugar portion.
 2. The antisense oligonucleotide derivative according toclaim 1, wherein said nucleotide analogue unit having a modified sugarportion has a structure represented by the following general formula

where B denotes a pyrimidine or purine nucleic acid base or an analoguethereof.
 3. The antisense oligonucleotide derivative according to claim1 or 2, which targets a 5′ untranslated region of hepatitis C virus RNA.4. The antisense oligonucleotide derivative according to claim 3,wherein said 5′ untranslated region is an internal ribosomal entry site(IRES).
 5. The antisense oligonucleotide derivative according to claim 1or 2, which targets a stem region of hepatitis C virus RNA.
 6. Theantisense oligonucleotide derivative according to claim 1 or 2, whichcontains any one of the following base sequences:CTTTGAGGTTTAGGATTCGTGCTCATG (SEQ ID NO: 1) AGGTTTAGGATTCGTGCTCATG (SEQID NO: 2) TAGGATTCGTGCTCATG (SEQ ID NO: 3) CCGGTTCCGCAGACCACTAT and (SEQID NO: 4) ACCCGGTCGTCCTGGCAATT. (SEQ ID NO: 11)


7. The antisense oligonucleotide derivative according to claim 1 or 2,containing an oligonucleotide derivative which hybridizes with DNA orRNA of a sequence complementary to any one of the following basesequences under stringent conditions: CTTTGAGGTTTAGGATTCGTGCTCATG (SEQID NO: 1) AGGTTTAGGATTCGTGCTCATG (SEQ ID NO: 2) TAGGATTCGTGCTCATG (SEQID NO: 3) CCGGTTCCGCAGACCACTAT and (SEQ ID NO: 4) ACCCGGTCGTCCTGGCAATT.(SEQ ID NO: 11)


8. An anti-hepatitis C virus agent containing the antisenseoligonucleotide derivative against hepatitis C virus RNA according toclaim 1 or 2, as an active ingredient.
 9. The antisense oligonucleotidederivative according to claim 3, which contains any one of the followingbase sequences: CTTTGAGGTTTAGGATTCGTGCTCATG (SEQ ID NO: 1)AGGTTTAGGATTCGTGCTCATG (SEQ ID NO: 2) TAGGATTCGTGCTCATG (SEQ ID NO: 3)CCGGTTCCGCAGACCACTAT and (SEQ ID NO: 4) ACCCGGTCGTCCTGGCAATT. (SEQ IDNO: 11)


10. The antisense oligonucleotide derivative according to claim 4, whichcontains any one of the following base sequences:CTTTGAGGTTTAGGATTCGTGCTCATG (SEQ ID NO: 1) AGGTTTAGGATTCGTGCTCATG (SEQID NO: 2) TAGGATTCGTGCTCATG (SEQ ID NO: 3) CCGGTTCCGCAGACCACTAT and (SEQID NO: 4) ACCCGGTCGTCCTGGCAATT. (SEQ ID NO: 11)


11. The antisense oligonucleotide derivative according to claim 5, whichcontains any one of the following base sequences:CTTTGAGGTTTAGGATTCGTGCTCATG (SEQ ID NO: 1) AGGTTTAGGATTCGTGCTCATG (SEQID NO: 2) TAGGATTCGTGCTCATG (SEQ ID NO: 3) CCGGTTCCGCAGACCACTAT and (SEQID NO: 4) ACCCGGTCGTCCTGGCAATT. (SEQ ID NO: 11)